The use of nucleic acid hybridization methods to detect disease states and infectious agents is a rapidly emerging technology (1). These methods have largely involved simple nonradioactive formats, aimed at achieving acceptance in the clinical laboratory (2,3). These assays use chemiluminescent or enzyme labels, or combinations of these to obtain the desired sensitivity (4). The rapid sample preparation offered by PCR offers the possibility of developing rapid and simple assays.
Nucleic Acid Amplification Processes
It is well known that a nucleic acid such as deoxyribonucleic acid (DNA) is able to serve as its own template during self-replication. It is also well known that a double stranded or duplex nucleic acid can be separated into its component single strands. These properties have been exploited to permit the in vitro amplification and modification of nucleic acid sequences by the polymerase chain reaction (PCR).
PCR is an in vitro, enzyme-based replication of nucleic acid sequences, using two oligonucleotide primers designed to hybridize to opposite strands and flank the region of interest on the target polynucleotide sequence. During repetitive cycles the nucleic acid is subjected to strand separation, typically by thermal denaturation, the primers are hybridized (by annealing if thermal cycling is used) to the single strand templates, and an enzyme such as DNA polymerase (DNA template to DNA primer extension) or reverse transcriptase (ribonucleic acid or "RNA" template to DNA primer extension or DNA template to DNA primer extension) extends the primers on the templates. Both of the strands (plus and minus), including newly synthesized strands are made available as templates for the extension of both primers respectively by the strand separation step. The result, with two primers, is an exponential increase (hence the term "chain reaction") in template nucleic acid copy number (both plus and minus strands) with each cycle, because with each cycle both the plus and minus chains are replicated. The nucleic acid duplex which results will have termini corresponding to the ends of the specific primers used. It is possible, by means of PCR, to amplify, detect, or otherwise modify a nucleic acid sequence in vitro.
The preparation of primers for PCR requires that the terminal sequences of the nucleic acid strands (both the plus and minus templates) to be amplified or detected, be known (5). The sequence information may be derived by direct sequencing of the terminals of the nucleic acid of interest, or by sequencing the terminal of a polypeptide and producing a corresponding copy oligonucleotide primer. The optimal primer size is typically about 20-30 bases in length, but workable primers may be smaller or larger in particular circumstances. As is well known, as primer size decreases, the likelihood that the primer will hybridize to an unplanned site on the sequence of interest increases. Unplanned hybridizations can lead to an interruption of amplification of the desired product and production of products having either a smaller size or an undesired primer insert. Thus, the selection of two optimal primers for PCR requires the avoidance of unplanned hybridization with the sequence of interest whenever practical.
The rational selection of primer sequence to avoid unplanned hybridizations is well known. Algorithms are known by which the artisan may compare proposed primer sequences to the entire template sequence (where known) and to any other sequences which are known to be present in an assay mixture.
The necessity for determining the terminal portion of the opposite strands of a nucleic acid sequence of interest and preparing two primers hybridizable thereto may be avoided by means of a universal primer. All DNA sequences present will receive a universal primer binding site and be amplified by the universal primer.
PCR amplification has been used to isolate new gene sequences from a polynucleotide sequence library. While new genes may also be isolated by means of a sufficiently complementary probe incorporating a portion of the sequence of the new gene, such probe isolation methods lack the sensitivity provided by PCR.
In the prior art assays based upon PCR, nucleic acid probes labeled at their 5' end have been used to hybridize to the nucleic acid analytes of interest. These probes occasionally enter into the polymerase chain reaction at their unlabeled 3' end resulting in spurious results. The prior art assays are also heterogeneous, i.e. separation assays. As such they are time consuming and the multiple wash steps involved introduce the possibility of contamination of the assay sample.
Detection of Labeled Nucleic Acid Sequence
Numerous methods and systems have been developed for the detection and quantitation of nucleic acid analytes of interest in biochemical and biological substances. Typically, the existence of a nucleic acid analyte of interest is indicated by the presence or absence of an observable "label" attached to a probe which binds to the analyte of interest. Of particular interest are labels which can be made to luminesce through photochemical, chemical, and electrochemical means.
"Photoluminescence" is the process whereby a material is induced to luminesce when it absorbs electromagnetic radiation. Fluorescence and phosphorescence are types of photoluminescence. "Chemiluminescent" processes entail the creation of luminescent species by chemical transfer of energy. "Electrochemiluminescence" entails creation of luminescent species electrochemically.
Electrochemiluminescent (ECL) assay techniques are an improvement on chemiluminescent techniques. They provide a sensitive and precise measurement of the presence and concentration of an analyte of interest. In such techniques, the incubated sample is exposed to a voltametric working electrode in order to trigger luminescence. In the proper chemical environment, such electrochemiluminescence is triggered by a voltage impressed on the working electrode at a particular time and in a particular manner. The light produced by the label is measured and indicates the presence or quantity of the analyte. For a fuller description of such ECL techniques, reference is made to PCT published application U.S. Ser. No. 85/02153 (W086/02734), PCT published application U.S. Ser. No. 87/00987 (W087/06706) and PCT published application U.S. Ser. No. 88/03947 (W089/04302). The disclosures of the aforesaid applications are incorporated by reference.
It is possible to carry out electrochemiluminescent assays with and without a separation step during the assay procedure, and to maximize the signal modulation at different concentrations of analyte so that precise and sensitive measurements can be made.
PCT published application number U.S. Ser. No. 89/04919 (W090/05301) teaches sensitive, specific binding assay methods based on a luminescent phenomenon wherein inert microparticulate matter is specifically bound to one of the binding reactants of the assay system. The assays may be performed in a heterogeneous (one or more separation steps) assay format and may also be used most advantageously in a homogeneous (nonseparation) assay format.
The luminescence arises from electrochemiluminescence (ECL) induced by exposing the label compound, whether bound or unbound to specific binding partners, to a voltametric working electrode. The ECL reactive mixture is controllably triggered to emit light by a voltage impressed on the working electrode at a particular time and in a particular manner to generate light.
U.S. patent application Ser. No. 267,509, now abandoned, and U.S. patent application Ser. No. 266,914, now abandoned, relate to preferred assay compositions. The disclosures of these applications are incorporate by reference.
U.S. patent application Ser. No. 267,234, now U.S. Pat. No. 5,061,445 and U.S. patent application Ser. No. 744,890, now U.S. Pat. No. 5,247,243 teach preferred apparatus for the conduct of ECL-based assays. U.S. patent application Ser. No. 652,427 describes preferred methods and apparatus for conducting ECL-based assays. The disclosures of all these applications, which are also incorporated by reference, permit the detection and quantitation of extremely small quantities of analytes in a variety of assays performed in research and clinical settings.